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The genetic diversity of plum pox virus strain M (PPV-M) was assessed by analyzing 28 isolates collected in 8 European countries. Two genomic fragments spanning the (Cter)P3-6K1-(Nter)CI coding region as well as the full coat protein coding region were sequenced directly from PCR products. Phylogenetic analysis showed that the geographical origin of the collected isolates was clearly associated with two different PPV-M clades. Moreover, the pattern of substitutions in the CP gene shed light on the evolutionary relationships between PPV-M and the recombinant strains PPV-Rec and PPV-T.

Genetic variability in modern crops is limited due to domestication and selection processes. Genetic variation in eight Bulgarian tomato varieties and breeding lines - variety Plovdivska karotina, variety IZK Alya, L21β, L53β, L1140, L1116, L975, L984 differing in their morphological and biochemical composition was assessed using a highly efficient and low-cost fluorescent simple sequence repeat /SSR/ genotyping platform. Genotyping was conducted with 165 publicly available microsatellite markers developed from different research groups under a number of projects in tomato - SOL Genomics SSRs, Kazusa TGS and TES, SLM, TMS and LEMDDNa among which only five - 3.03% failed to amplify the expected PCR fragments. Of the remaining markers, 81 - 50.62% were polymorphic in the whole collection of eight genotypes. Among the marker groups used, SLM markers were most polymorphic, followed by TMS and SOL Genomics SSR markers. The total number of amplified alleles was 299, with a mean of 1.869; and the average polymorphic information content - PIC was 0.196. The genetic diversity within the collection was relatively low - 0.2222. Neis genetic distance varied from 0.0953 to 0.3992. Cluster analysis using the un-weighted pair group method with arithmetic mean - UPGMA method indicated that the studied tomato genotypes are grouped in four main clusters, which is to some extent consistent with the morpho- and hemo-types of the studied tomatoes. Variety IZK Alya - cherry type and two of the breeding lines - L1140, L1116 formed three separate and more distant clusters. The fourth cluster includes the other five genotypes. The observed grouping of these genotypes in two sub-clusters reflects their similar morphological and biochemical composition. The genetic distance information from this study might be useful for further implementation of breeding strategies and crosses among these inbred lines.

Insertion mutant collections are powerful tools for genetic studies in plants. Although large-scale insertional mutagenesis using T-DNA is not feasible in legumes, the Tnt1 tobacco retrotransposon can be used as a very efficient mutagen in the Medicago truncatula R108 genotype. In this article, we show that Tnt1 can also be exploited to create insertional mutants via transformation and/or regeneration in the reference cultivar Jemalong. Tnt1 insertional mutagenesis in Jemalong following Agrobacterium tumefaciens-mediated transformation was found to be very efficient, with an average of greater than 15 insertions/line. In contrast, regeneration using low-copy transgenic starter lines resulted in a highly variable rate of new Tnt1 insertions. With the goal of increasing the number of additional Tnt1 insertions during regeneration of starter lines, we have compared the insertion frequencies for a number of different regeneration protocols. In addition, we have been able to show that sucrose-mediated osmotic shock preceding regeneration significantly increases the transposition frequency. Under optimal conditions, 95% of the regenerated Jemalong plants possess new insertions.

The Tnt1 retrotransposon of tobacco (Nicotiana tabacum) has proven to be a very efficient mutagen for the model legume Medicago truncatula ecotype 108 and cultivar Jemalong 2HA and for economically important plants, such as soybean and potato. In this study, the activity of Tnt1 in the model legume Lotus japonicus L. was tested. First, a new regeneration and transformation protocol was developed for L. japonicus that represents a new tool for legume mutagenesis and reverse genetics. Using this protocol, the Tnt1 retrotransposon was introduced into L. japonicus by Agrobacterium tumefaciens-mediated transformation, and primary transgenic lines, named starter lines, were constructed. In vitro regeneration via indirect somatic embryogenesis using starter lines harboring two to eight copies of the transgene resulted in new Tnt1 transposition events. The Tnt1 retrotransposon remained inactive during plant growth and in the T-1 progeny, indicating that it is well suited for insertional mutagenesis in L. japonicus.

In 2014, a total of 108 symptomatic watermelon samples were collected in Greece and analyzed by DAS-ELISA using polyclonal antisera (INRA, Montfavet) against the most common aphid-transmitted viruses. Cucumber mosaic virus (CMV) was detected in eight of 108 plants tested. Five of these plants collected at Lesvos were also infected with Watermelon mosaic virus (WMV), whereas three plants from Prohoma had single infections. Five samples with severe stunting and yellowing collected at Asenovgrad (Bulgaria) in 2013 and tested by DAS-ELISA for the above viruses hosted CMV and Cucurbit aphid-borne yellows virus (CABYV). To confirm CMV presence in watermelon, a nested RT-PCR assay was carried out first with the degenerated primers CMVup624a (5′-ATGGACAAATCTGRATC- 3′) and CMVdo1244a (5′-TGRTGCTCRAYGTCKACATGA-3′) followed by CMVup624b (5′-GGACAAA TCTGRA TCTCCCAA TGC-3′) and CMVdo1244b (5′- TGCTCRAYGTCRACATGAAG-3′) that amplify a 622 bp region from the viral coat protein gene. Total RNA (Chatzinasiou et al., 2010, method A) extracted from all ELISA- positive watermelon samples from both countries and from two healthy watermelon plants was used as template in nested RT-PCR. A product of the expected size was amplified from all serologically CMV-positive samples, but not from the healthy ones. The Greek CMV isolates from Lesvos (LN810059) and Prohoma (LN810060) showed 99% nucleotide sequence identity with potato (AB448694) and tomato (EF153734) CMV isolates from Syria and India, while the Bulgarian isolate (LN810058) showed 99% nucleotide sequence identity with an Indian cucumber isolate (JF279608). Phylogenetic analysis indicated that the Greek CMV isolate (LN810059) belongs to sub-group IA, while the Bulgarian isolate and a Greek isolate (LN810060) belong to sub-group IB. To our knowledge this is the first report of CMV infecting watermelons in Greece and Bulgaria.

Summer snowflake /Leucojum aestivum L./ is a medical bulbous plant which belongs to family Amarillydaceae. It is wild-spread as natural populations in Bulgaria and a valuable source of galantamine. A few virus and fungal diseases have been described on plants of family Amaryllidaceae but only fungal pathogens on summer snowflake. This paper is the first report of Serratia plymuthica and Stenotrophomonas maltophilia as causal agents of bulb decay of summer snowflake. To our knowledge, this is also the second report of phytopathogenic isolates of the identified two species. The bacteria were isolated from plants in the region of Tutrakan close to the Danube River where pathogenic changes in plant tissues of bulbs of summer snowflake were detected in 2006. Pathogenicity to the natural plant host was confirmed by artificial inoculation. Identification was carried out by biochemical properties and sequence analysis. New hosts upon artificial inoculation of S. maltophilia and S. plymuthica are onion, hyacinth, tulip, narcissus, and crocus. The established bacterial species are known inhabitants of the soil and rhizosphere, contaminants of food and water, human infectious agents, and represent a case, similar to Serratia marcescens, when a species can comprise saprophytic, plant, and human strains simultaneously.

The Tnt1 retrotransposon of tobacco (Nicotiana tabacum) has proven to be a very efficient mutagen for the model legume Medicago truncatula ecotype 108 and cultivar Jemalong 2HA and for economically important plants, such as soybean and potato. In this study, the activity of Tnt1 in the model legume Lotus japonicus L. was tested. First, a new regeneration and transformation protocol was developed for L. japonicus that represents a new tool for legume mutagenesis and reverse genetics. Using this protocol, the Tnt1 retrotransposon was introduced into L. japonicus by Agrobacterium tumefaciens-mediated transformation, and primary transgenic lines, named starter lines, were constructed. In vitro regeneration via indirect somatic embryogenesis using starter lines harboring two to eight copies of the transgene resulted in new Tnt1 transposition events. The Tnt1 retrotransposon remained inactive during plant growth and in the T-1 progeny, indicating that it is well suited for insertional mutagenesis in L. japonicus.